Our long-term goal is to understand the mechanisms that underlie the development, maintenance and degeneration of the mechanosensory hair bundle. Motivated by the fact that a third of all known deafness genes encode hair bundle proteins, we previously used a mass spectrometry based strategy to characterize the hair bundle proteome. This approach proved to be a powerful complement to traditional genetics strategies, leading to the discovery of novel deafness genes previously undetected using genetic strategies alone. For further analysis, we focused on proteins for which the corresponding gene locations overlap with unresolved human deafness loci. One such protein is XIRP2 (for xin actin-binding repeat containing protein 2). In this project, we are testing the hypothesis that XIRP2 is a novel hair cell protein required for long-term stability of stereocilia and the hair cell/supporting cell junctions, with the consequence that XIRP2 deficiency causes hair cell degeneration and progressive hearing loss. Preliminary studies demonstrated that different XIRP2 splice forms are present in the hair cell, displaying distinct localizations in the stereocilia and pericuticular adherens junctions, respectively. To explore the role of XIRP2 for hearing function, we used the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas technology to generate transgenic mice with a functional null mutation in the Xirp2 gene and found evidence for high-frequency hearing loss. In Specific Aim 1, we will investigate the hearing loss phenotype in Xirp2 null mice in detail. Furthermore, we will test whether XIRP2 deficiency renders the hair cell more sensitive to mechanical stress, by testing the sensitivity of Xirp2 null mice to noise-induced hearing loss. Preliminary studies suggested that the stereocilia and the adherens junctions harbor distinct XIRP2 isoforms, implying that defects in either hair cell structure could underlie the observed hearing loss in Xirp2 null mice. n Aim 2 and 3, we will therefore use transgenic mice with isoform specific deletions in the Xirp2 gene, to specifically address their contribution to hair cell degeneration and hearing loss. Our hypothesis that XIRP2 is required for long-term maintenance of hair cell structures dovetails well with its known role in cardiac muscle, where it is involved in the maintenance of the sarcomeric Z-line. XIRP2's role in the hair cell is likely to be distinct from and more complex than its functon in the heart: the hair bundle harbors a novel isoform with vastly different protein domain structure, rendering it practically an unknown protein. Significant for human hearing health, understanding the role of XIRP2 in hair cell degeneration is expected to shed light on mechanisms by which subtle defects caused by genetic and environmental factors can compromise the structural integrity of hair cell structures, significant for understanding the mechanistic basis of age and noise-induced hearing loss. Finally, the chromosomal locus of the XIRP2 gene overlaps with the human deafness loci DFNB27 and DFNA16, opening up the prospect of identifying the causative mutation for two human deafness phenotypes.